1. Field of the Invention
This invention provides methods for identifying polypeptides which selectively coordinate to metals. This invention also provides chemosensors comprising polypeptides which coordinate to metals and methods for detecting the presence of metals using these chemosensors.
2. Background of the Invention
Fluorescent indicators have revolutionized the process of quantifying metal cations in aqueous media, and in particular within biological samples. The success of fluorescent indicators for the intracellular measurement of sodium and potassium (Minta, A.; Tsien, R. Y., J. Biol. Chem. 1989, 264:19449-19457), calcium (Tsien, R. Y., Biochemistry 1980, 19:2396-2404; Grynkiewicz, G., et al., J. Biol. Chem. 1985, 260:3440-3450), magnesium (Raju, B., et al., Am. J. Physiol. 1989, 256: C540), and pH (Bassnett, S., et al., Am. J. Physiol. 1990, 258: C171-C178; Rink, T. J., et al., Cell Biol. 1982, 95:189-196) is well known. Due to the success of these agents, the design and production of fluorescent chemosensors for other species continues to be an active area of interest.
The central problem in the production of new fluorescent sensors for the detection of metal cations lies in selectivity (Czarnik, A. W., Supramolecular Chemistry, Fluorescence, and Sensing. In Fluorescent Chemosensorsfor Ion and Molecule Recognition; Czarnik, A. W.; ACS, Washington, D.C., 1993; pp. 1-9). In fact, there are successful intracellular fluorescent probes only for the divalent cations Mg.sup.2+ and Ca.sup.2+, which are present at the highest concentration within the cell. For example, the concentration of ionized Zn.sup.2+ within a cell or in sea water may be as much as 10.sup.6 fold less concentrated than that of Mg.sup.2+ or Ca.sup.2+ (Bruland, K. W., Trace Elements in Sea Water. In Rilet, J. P.; Chester, R.; Academic Press, London, 1975; pp. 157-220; Frausto da Silva, J. J. R.; Williams, R. J. P., The Biological Chemistry of the Elements: The Inorganic Chemistry of Life; Clarendon Press: New York, 1993.). Thus, the fluorescent indicator fura-2 may bind Zn.sup.2+ with greater affinity than Ca.sup.2+, but remains a cellular probe for free calcium. In order to prevent spurious cross-talk, the relative affinity of the sensor for the ion of interest must exceed the cumulative concentration excess imposed by all other competing species. Typically, this difficulty has been addressed by the exploitation of proteins for their unmatched selectivity in binding small molecules (Giuliano, K. A., et al., Ann. Rev. Biophys. Biomol Struct. 1995, 23:405-434). Thus biological signal transducers, i.e. "biosensors," have been devised from existing proteins for the divalent cations of zinc (Thompson, R. B., Anal. Chem. 1993, 65:730-734; Thompson, R. B.; Patchan, M. W., Anal. Biochem. 1995, 227:123-128), mercury (Virta, J.; Lampinen, J.; Karp, M., Anal. Chem. 1995, 67:667-669), as well as copper and cobalt (Thompson, R. B., et al., Biosensors & Bioelectronics 1996, 11:557-564), and even organic molecules such as cAMP (Adams, S. R., et al., Nature 1991, 694-697).
Nonetheless, the need for new chemosensors for these and other analytes continues to exist (Czarnik, A. W., Chem. Bio. 1995, 2:423-428). Although the analyte binding selectivity which may be obtained with a biosensor is remarkable, the complexity of a large biomolecule can impose greater design constraints relative to an abiotic sensing molecule. For example, proteins typically lack the fluorescence characteristics of a useful sensor, and thus a strategy involving affinity labeling or an auxiliary diffusible fluorophore is required for signal transduction. In this light, the production of a purely synthetic chemosensor is desirable as there is greater flexibility for systematic variation of the analyte-binding and fluorescent moieties of the sensor. The production of peptidyl motifs with tunable metal binding properties (Cheng, R. P., et al., J. Am. Chem. Soc. 1996, 118:11349-11356), as well as those with novel fluorescent signaling capabilities (Torrado, A.; Imperiali, B., J. Org. Chem. 1996, 61:8940-8948) highlights the applicability of this technique.
Previously, the present inventor has investigated the production of zinc-sensing fluorosensors using a hybrid approach (Walkup, G. K.; Imperiali, B., J. Am. Chem. Soc. 1996, 118:3053-3054). By exploiting the selective metal binding properties of the zinc finger domains (Berg. J. M.; Merkle, D. L., J. Am. Chem. Soc. 1989, 111:3759-3761), the advantageous aspects of abiotic chemosensors and biosensors have been combined within a synthetic polypeptide architecture. Zinc finger peptides bind divalent zinc avidly, with dissociation constants as low as 5.7 pM reported for the peptide.multidot.Zn.sup.2+ complex (Krizek, B. A.; Merkle, D. L.; Berg, J. M., Inorg. Chem. 1993, 32:937-940), and with great selectivity (Krizek, B. A.; Berg, J. M., Inorg. Chem., 1992, 31:2984-2986).
A single zinc finger domain is 25-30 residues in length and may be described by the consensus sequence EQU (F/Y)-X-C-X.sub.2-4 -C-X.sub.3 -F-X.sub.5 -L-X.sub.2 -H-X.sub.3-5 -H-X.sub.2-6
where X is any amino acid (Berg, J. M., Acc. Chem. Res. 1995, 28:14-19; Klug, A.; Schwabe, J. W. R., FASEB J. 1995, 9:597-604). Importantly, peptides of these lengths are synthetically accessible by chemical techniques. Furthermore, zinc fingers have been shown to undergo a reversible metal-induced conformational change (Eis, P. S.; Lakowicz, J. R., Biochemistry 1993, 32:7981-7993; Frankel, A. D., et al., Proc. Natl. Acad Sci. U.S.A. 1987, 84:4841-4845), which nucleates a cluster of hydrophobic residues (underlined above).
The present inventor reported the synthesis and characterization of a fluorescent synthetic peptidyl chemosensor for divalent zinc, patterned after the zinc fingers (Walkup, 1996, supra; Walkup, G. K., Imperiali, J. Am. Chem. Soc. 1997, in press). An aromatic residue of the hydrophobic cluster of the parent sequence was replaced with a derivative of .beta.-amino alanine, incorporating an orthogonally protected side-chain amine. At the completion of the peptide synthesis, this residue was selectively deprotected, then coupled with a variety of amine-reactive fluorophores to produce a selectively labeled fluorescent peptide. Deprotection and cleavage of the peptide from the synthesis resin afforded the completed chemosensor. The microenvironment experienced by the fluorophore-bearing residue changed upon peptide.multidot.Zn.sup.2+ complex formation, resulting in enhanced fluorescence. In addition, the subsequent report of another sensor developed along similar lines was noted (Godwin, H. A.; Berg, J. M., J. Am. Chem. Soc. 1996, 118:6514-6515), but which uses two fluorophores and a resonant energy transfer mechanism for signal transduction.
Both of these sensors are capable of quantifying nanomolar concentrations of Zn.sup.2+, but are susceptible to oxidation through the formation of an intramolecular disulfide bond and are thus incompatible with aqueous oxidants including oxygen and redox active ions such as Cu.sup.2+. This may not be problematic in the reducing environment of a cell, but application of these sensors toward the measurement of environmental samples would be precluded.
Despite these and other advances, chemosensors with broader applicability, increased selectivity and increased sensitivity are desirable.